Petroleum derived fuels have been the primary source of energy for over a hundred years. Petroleum is formed over millions of years in nature and is a non-renewable source of energy. A significant amount of research in biofuels has been ongoing for decades. Within this field, ethanol has been studied intensively as a gasoline substitute. However, the efficiency of ethanol as a fuel remains debatable. (Pimentel, Natural Resources Research (2005) 14:65; Farrell et al., Science (2006), 311:506).
The alcohol 3-methyl-butanol has been demonstrated to be a potential biofuel in both spark-ignition and homogenous charge compression ignition (HCCI) engines. The compound can be synthesized from the isoprenoid pathway by converting the isoprenoid intermediate isoprenoid precursors isopentyl pyrophosphate (IPP) to dimethylallyl diphosphate (DMAPP), DMAPP to 3-methyl-2-butenol, and 3-methyl-2-butenol to 3-methyl-butanol (FIG. 1). The enzymes required for performing each individual step are: a phosphatase to convert IPP to 3-methyl-3-butenol, a phosphatase to convert DMAPP to 3-methyl-2-butenol, and a reductase to convert 3-methyl-2-butenol to 3-methyl-butanol. The enzymes can be expressed to obtain all of these three 5-carbon alcohols (see, e.g., U.S. Pat. No. 7,985,567, which is incorporated by reference). The conversion of IPP to DMAPP requires expression of an IPP isomerase.
The present invention relates to compositions and methods for expressing IPP isomerase as a fusion protein linked to a phosphatase to increase the production of 3-methyl-2-butenol from IPP and thus provides a method for increasing the production of 3-methyl 2-butanol for biofuel production.